JPH0475465B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to methods and reagents for the determination of ligands in biological fluids such as serum, plasma, spinal fluid, amniotic fluid and urine. The present invention also relates to a new class of fluorescein derivatives that can be used as reagents in fluorescence polarization immunoassays. Competitive binding immunoassays for measuring ligands are based on competition between a ligand in a test sample and a labeled reagent called a tracer for a limited number of receptor binding sites on an antibody specific for the ligand and reagent. The concentration of ligand in the sample determines the amount of tracer that specifically binds to the antibody. The amount of tracer-antibody conjugate produced can be measured quantitatively and is inversely proportional to the amount of ligand in the test sample. Fluorescent polarization is based on the principle that when a fluorescently labeled compound is excited by linearly polarized light, it emits fluorescent light with a degree of polarization that is inversely proportional to its rotation rate. Therefore, when a molecule such as a fluorescently labeled tracer-antibody conjugate is excited by linearly polarized light, the emitted light is very small because the light is absorbed and the fluorophore is restrained from rotation during the emitted time. remains polarized. When a “free” tracer compound (i.e., not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate, causing the molecules to become more disorderly. The generated light is then depolarized. Fluorescence polarization therefore provides a means for measuring the amount of tracer-antibody conjugate produced in competitive binding immunoassays. A variety of fluorescently labeled compounds are known in the art. U.S. Pat. No. 3,998,943 discloses fluorescently labeled insulin derivatives using fluorescein isothiocyanate (FITC) as the fluorescent label and fluorescently labeled morpholin derivatives using 4-aminofluorescein hydrochloride as the fluorescent label. The production of each derivative is described. Carboxyfluorescein has also been used for analytical measurements. RC Chin Analytical Letters, 10 , 787
(1977) describe the use of carboxyfluorescein, which exhibits phospholipase activity. Carbofluorescein is contained in lecithin intracellular fat particles and only fluoresces when released by hydrolysis of lecithin. 1981
U.S. Patent Application No. 329,974, filed Nov. 11, discloses a class of carboxyfluorescein derivatives in which carboxyfluorescein is attached directly to a ligand-congener useful as a reagent in fluorescence polarization immunoassays. are doing. The present invention provides a sample with the formula: (T in the formula is

[Formula] represents a group, n is an integer from 1 to 8, and R is a reactive primary bonded to the carbonyl carbon of the group represented by T.
or a secondary amino group and at least one epitope in common with the above-mentioned ligands such that they can be specifically identified by a common antibody.
of the concentration of said ligand in the sample after mixing with a biologically acceptable salt of a tracer having a molecule (representing a ligand congener with said ligand) and an antibody capable of specifically identifying said ligand and said tracer. A method of measuring the ligand in a sample consists of determining the amount of tracer antibody conjugate by fluorescence polarization as a measure. As used herein, "ligand" refers to a molecule, particularly a low molecular weight hapten with a single reactive amino group from which a binding protein, usually an antigen, is obtained or produced. This hapten is a protein-free compound and generally has a low molecular weight, so it does not produce antibodies when injected into an animal, but it is reactive with antibodies. Antibodies to haptens are generally produced by first conjugating the hapten to a protein and injecting the conjugated product into an animal. The resulting antibodies can be isolated using standard antibody separation methods. The ligands that can be measured by the method of the invention span a wide molecular weight range. High molecular weight ligands can also be measured, but generally 50 to 4000
The method of the present invention may be used to measure low molecular weight ligands in the range of . More preferred is a ligand assay with a molecular weight range of 100 to 2000. Representative ligands that can be measured by the method of the present invention include estriolestrone, estradiol,
cortisol, testosterone, progesterone,
Steroids such as deoxycholic acid, lithocholic acid and their ester and amide derivatives;
Vitamins such as B-12, fluoric acid; prostaglandins such as thyroxine, triiodothyroxine, histamine, serotonin, PGE, PGF, and PGA; anti-asthmatic agents such as theophylline, antineoplastics such as doxorubicin and methotrexate. Plastic agents; disopyramide, lidocaine, procainamide, propranolol, quinidine, N-
antiarrhythmic agents such as acetyl-procainamide;
Anticonvulsants such as phenobarbital, fenithion, primidone, valproic acid, carbamazepine and esosuximide; antibiotics such as penicillin, cephalospirin and vancomycin; antiarthritic agents such as salicylate; nortriptyline, amitriptyline, imipramine and desipramine and their metabolites. Further, ligands that can be measured by the method of the present invention include morphine, heroin, hydromorphone, oxymorphone, metapone, codeine, hydrocodone, dihydrocodeine, dihydrohydroxycodeinone,
There are drugs and their metabolites that have harmful effects, such as phorcozine, dextromethorphan, phenazosine, and deonine. As used herein, a ligand homologue refers to one or more determinants that can compete with the ligand for receptor binding position or a substantial portion that determines epitopic position in the same spatial location as the ligand. and a monovalent or polyvalent group with a polar configuration. A characteristic of this ligand homolog is that it has sufficient structural similarity to the ligand in question to be recognized by antibodies of the ligand. Often the ligand homolog has the same or substantially the same structure and charge distribution (spatial and polar configuration) over a large portion of the molecular surface as the ligand in question. Frequently, the binding site of the hapten is the same as in the production of antigens for antibody generation, such as those used for binding to the ligand, so that the same portion of the ligand homolog that serves as the template for the antibody is the same as the ligand homolog in the tracer. will be exposed by the body. The type of ligand homolog generally represented by R is determined by removal of the hydrogen atom bonded to the reactive amine (primary or secondary) or by removal of the amino group.

[Formula] is derived from the corresponding ligand by forming an amino derivative of the ligand that replaces one or more atoms originally present in the ligand at the bonding position to the carbonyl carbon of the group represented by T. be done. Examples of ligands whose removal of hydrogen from an active amino group produces a ligand analog represented by R include, for example, procainamide, thyroxine and quinidine.
Examples of ligands whose amino derivatives are useful as ligand congeners include theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide, digoxin, chloramphenicol, salicylate, acedaminophene, carbamazepine, These include desipramine and nortriptyline. Additionally, the ligand can be structurally modified by the addition or deletion of one or more reactive groups to produce ligand homologues that retain the epitope position necessary to bind to the antibody. However, this modified ligand analog is attached to the carbonyl carbon of the group represented by T through the imino group. Preferably, n in the group represented by T is 2 to 4. The tracers of the present invention are generally in equilibrium between their acid and ionized states and are effective in the ionized state in the methods of the present invention. The invention therefore also encompasses tracers in either the acid or ionized state, and conveniently the tracer in its ionized state is present in the form of a biologically acceptable salt. As used herein, "biologically acceptable salts" refer to salts such as sodium, potassium, ammonium, etc., which cause the tracers of the invention to be in an ionized state when used in the methods of the invention. Generally, the tracer of the present invention is in solution as a salt, the specific salt being obtained from the buffer used, i.e. in the presence of a sodium phosphate buffer, and the tracer of the present invention is generally present in its ionized state as a sodium salt. . According to the method of the invention, a sample containing the ligand to be measured is mixed with a biologically acceptable salt of a tracer having the formula () and an antibody specific for the ligand and the tracer. The ligand and tracer present in the sample compete for defined antibody positions, producing ligand-antibody and tracer-antibody complexes. By holding the concentrations of tracer and antibody constant, the ratio of ligand-antibody conjugate to tracer-antibody conjugate produced is directly proportional to the amount of ligand present in the sample. Therefore, by exciting the mixture with polarized light and measuring the polarized light of the fluorescent light generated by the tracer and tracer antibody complex, the amount of ligand in the sample can be quantitatively measured. Theoretically, the fluorescence polarization of tracers that do not complex with antibodies is small and close to zero. The tracer-antibody complex formed when complexed with a specific antibody rotates the antibody molecule more slowly than a relatively small tracer molecule, resulting in an increase in polarization. Therefore, when the ligand competes with the tracer for antibody position, the observed polarization of the fluorescence of the tracer-antibody complex will be between the values of the tracer and the tracer-antibody complex. If the sample contains a high concentration of ligand, the observed polarization value will be close to that of free ligand, ie small. If the sample has a small concentration of ligand, the polarization value will be close to that of the bound ligand, ie, it will be high. By sequentially exciting the reaction mixture of an immunoassay with vertically polarized light and then with horizontally polarized light and analyzing only the vertical component of the emitted light, the polarization of the fluorescent light in the reaction mixture can be accurately determined. An accurate relationship between polarization and concentration of the ligand to be measured can be established by measuring the polarization value of a corrector with known concentration.
Ligand concentration can be extrapolated from the standard curve created in this way. The pH in carrying out the method of the invention is a pH sufficient to place the tracer with formula () in its ionized state.
It has to be. The PH is about 3-12, usually about 5-10, most preferably about 6-9.
Various buffers are used to maintain the pH as above during the test. Typical buffers include borate, phosphate, carbonate, Tris, barbital, and the like.
The particular buffer used is not critical to the invention, but
For each test, a specific buffer is preferred, taking into consideration the antibody used and the ligand to be measured. The cationic portion of the buffer will generally determine the cationic portion of the tracer salt in solution. The method of the invention is carried out at moderate temperatures, preferably at a constant temperature. The temperature is usually between 0 and 50°C, preferably about 15°C.
The temperature ranges from 40°C to 40°C. The ligand concentrations that can be analyzed are generally about 10 ^-2 to
10 ^-13 M, more commonly varying from about 10 ^-4 to 10 ^-10 M. Highly concentrated ligands can be tested after diluting the original sample. In addition to the concentration of ligand to be analyzed, consideration of whether the test will be conducted qualitatively, semi-quantitatively, or quantitatively, the equipment used, and the characteristics of the tracer and antibody will usually determine the tracer and antibody concentrations used. Probably. Although the concentration of the ligand in the sample determines the concentration range of the other reagents, ie, tracer and antibody, individual reagent concentrations are usually determined empirically to optimize test sensitivity. Tracer and antibody concentrations can be readily determined by those skilled in the art. Preferred tracers of the invention are characterized as derivatives of 5-carboxyfluorescein or 6-carboxyfluorescein or mixtures thereof and have the formula: It is represented by. The examples set forth below are intended to demonstrate to those skilled in the art how to make specific tracers within the scope of the invention, and are not intended to limit the invention. [CF] in the structural formula showing the compound produced in the following examples is the formula: represents the part with . where the carbonyl carbon is attached at the 5 or 6 position depending on whether the starting material in the example is 5-carboxyfluorescein, 6-carboxyfluorescein or a mixture thereof. Example 1 Preparation of N-hydroxysuccinimide active ester of carboxyfluorescein A solution of 83 mg (0.22 mmol) of 6-carboxyfluorescein in 2 ml of dimethylformamide contains 28 mg (0.24 mmol) of N-hydroxysuccinimide and N , 55 mg (0.27 mmol) of N'-dicyclohexylcarbodiimide were added. The reaction mixture was stirred under argon atmosphere at 0°C for 1 hour and then at 4°C for 16 hours to give the formula: An N-hydroxysuccinimide active ester of carboxyfluorescein was obtained. Example 2 5.85 g of 5-aminovalerianic acid in 100 ml of 2% aqueous sodium hydroxide solution and 100 ml of isoxane
A solution containing 10.9 g (0.05 mol) of di-tert-butyl dicarbonate in 40 ml of dioxane was added dropwise to the solution containing (0.05 mol) di-tert-butyl dicarbonate. The reaction mixture was stirred for 18 hours and then acidified to PH3 using 1N HCl. The mixture was extracted three times with dichloromethane, the organic layers were combined, washed with water, dried over sodium sulfate, and 5-(t-
10.1 g (93.5% yield) of white crystalline solid of butoxycarbonylamino)valerianic acid were obtained. 0.434 g (0.002 mol) of 5-(t-butoxycarbonylamino)valerianic acid contains 0.412 g (0.002 mol) of N,N'-dicyclohexylcarbodiimide and 0.25 g (0.0022 mol) of N-hydroxysuccinimide in 3 ml of dichloromethane. The solution was added with stirring and reacted for 18 hours to form an oily residue.
N-hydroxysuccinimide active ester of (t-butoxycarbonylamino)valerianic acid was obtained. 1.95 g (0.0022 mol) of L-thyroxine sodium salt pentahydrate in 30 ml of methanol to an oily residue.
A solution containing was added. After allowing the reaction to proceed for 18 hours, the reaction mixture was transferred to an ion exchange resin tube (Bio−Rad AG 50W−X8H ⁺ type).
Elute with methanol. Concentrate the eluent in vacuo and use the formula: 1.96 g (90% yield) of intermediate with 0.125 g (0.00015 mol) of the intermediate was treated with 2.0 ml of trifluoroacetic acid for 30 minutes, evaporated under reduced pressure to remove the trifluoroacetic acid, and the residue was dissolved in 1.5 ml of N,N'-dimethylformamide. The resulting solution was adjusted to basic pH with triethylamine. 75 mg (0.000159 mol) of N-hydroxysuccinimide active ester of carboxyfluorescein was added to the resulting mixture.
The reaction was allowed to proceed for 18 hours. The reaction mixture was precipitated by addition of diethyl ether, and the precipitate was purified by preliminary reverse-phase TLC using a methanol:water:acetic acid (75:25:0.5) mixture to give the formula: 0.071 g of a thyroxine-6-carboxyfluorescein conjugate having the following properties was obtained as an orange solid. Example 3 To a solution containing 10 g (0.1122 mol) of β-alanine in 100 ml of a 1:1 dioxane:water mixture containing 4.5 g (0.1125 mol) of sodium hydroxide, 40 g of dioxane was added.
26.95g di-t-butyl dicarbonate in ml
(0.1235 mol) was added dropwise. After stirring the reaction mixture for 16 hours, the pH was acidified to 3 with 1NHCl and extracted three times with dichloromethane. The organic layers were combined, washed with dilute hydrochloric acid solution, dried over magnesium sulfate, and washed with 3-t
-butoxycarbonylamino)propionic acid 17
g (93.5% yield). 2.1 g of 3-(t-butoxycarbonylamino)propionic acid dissolved in 25 ml of methylene chloride
(0.010 mol) of the solution were added 1.34 g (0.0116 mol) of N,N'-dicyclohexylcarbodiimide and 2.62 g (0.0127 mol) of N-hydroxysuccinimide. The reaction was allowed to proceed for 20 hours at room temperature under an argon atmosphere, yielding an oily residue which was redissolved in methylene chloride. The liquid was concentrated in vacuo to obtain a white solid of N-hydroxysuccinimide active ester of 3-(t-butoxycarbonylamino)propionic acid. In a solution containing 0.5 g (0.0006 mol) of L-thyroxine sodium salt pentahydrate in 2 ml of methanol, 3-
N-hydroxysuccinimide active ester of (t-butoxycarbonylamino)propionic acid 0.24
g (0.0008 mol) was added. As the reaction proceeded, a residue was formed, and upon completion of the reaction, 1N NaOH was added to dissolve the residue. The reaction product was purified using a silica gel tube eluting with a 1:4 mixture of methanol:methylene chloride. Concentrate the eluate and use the formula: We obtained an intermediate with . A solution of 0.2 g (0.00021 mol) of the intermediate in a saturated solution of dioxane containing hydrogen chloride was stirred at room temperature for 2 hours. Dioxane: The residue obtained by removing hydrochloric acid in vacuo is diluted with 1.5% N,N'-dimethylformamide.
It was dissolved into ml. The pH of the resulting solution was adjusted to neutral using triethylamine. Add succinimide active ester of 6-carboxyfluorescein to the resulting mixture.
107 mg (0.00022 mol) was added. The reaction was allowed to proceed for 16 hours under an argon atmosphere to give a crude product which was purified by preliminary reversed phase TLC using methanol:
Formula using a water:acetic acid (70:30:0.4) mixture: 10.0 mg (yield 4%) of a thyroxine-6-carboxyfluorescein conjugate having the following properties was obtained. Example 4 6-aminocaproic acid 10g (0.07623 mmol)
While constantly stirring the solution containing sodium hydroxide.
Dioxane containing 3.05g (0.07625mmol):
Added to a mixture containing 200 ml of a 1:1 mixture of water. A solution containing 16.54 g (0.07623 mol) of di-tert-butyl dicarbonate in 80 ml of dioxane was added dropwise to the resulting solution. The reaction mixture was stirred for 16 hours and then acidified to pH 3 with 1N hydrochloric acid. Dilute this solution with dichloromethane for 3
Extracted twice, the organic layers were combined and concentrated in vacuo, and the residue was dissolved in methylene chloride. The methylene chloride solution was washed with dilute hydrochloric acid, extracted with saturated sodium bicarbonate solution, and the aqueous layers were combined. The aqueous layer was adjusted to pH 3 with 1N hydrochloric acid and then extracted with methylene chloride. The methylene chloride extract was dried over magnesium sulfate and concentrated in vacuo to give 13 g of 5-(t-butoxycarbonylamino)caproic acid (yield
75%). 5-5 at room temperature under an argon atmosphere in 10 ml of a 1:1 mixture of methylene chloride:dimethylformamide.
(t-butoxycarbonylamino)caproic acid 1.0
g (0.00432 mol) was dissolved. Add N- to the solution
Hydroxysuccinimide 0.55g (0.00478mol)
Then 1.07 g (0.00519 mol) of N,N'-dicyclohexylcarbodiimide was added. After the reaction was allowed to proceed for 20 hours, the filtrate was filtered through Celite, the filtrate was concentrated, and the residue was redissolved in methylene chloride. The methylene chloride solution was overconcentrated to give a white solid. this solid
0.58g (0.00177mol) in 4ml of methanol
-Thyroxine sodium salt pentahydrate 0.5g
(0.00056 mol). This was stirred at room temperature under an argon atmosphere for 3 hours and then concentrated to give the formula: The intermediate was obtained as a tan powder with . 0.10 g (0.0001 mol) of the intermediate in methylene chloride:
A 1:1 mixture of trifluoroacetic acid was dissolved at 0° C. under an argon atmosphere. After 45 minutes, methylene chloride and trifluoroacetic acid were removed in vacuo, and the residue was dissolved in 1 ml of dimethylformamide, and the solution was adjusted to pH 8 with triethylamine. 0.38 g of 6-carboxyfluorescein dissolved in 1 ml of dimethylformamide
The above solution was added to a liquid containing 6-carboxyfluorescein imidizolide prepared by reacting (0.0001 mol) with 0.016 g (0.0001 mol) of 1,1'-carbonyldiimidizole. The reaction mixture was stirred at room temperature under argon for 4 hours to yield the crude product. This was purified by reverse phase thin layer chromatography and methanol:water:acetic acid (70:30:
0.4) Formula using a mixture: 0.054 g (yield: 43%) of thyroxine-6-carboxyfluorescein conjugate having the following properties was obtained. Example 5 N in 2 ml of N,N'-dimethylformamide,
N'-dicyclohexylcarbodiimide 0.353g
(0.0017 mol) and N-hydroxysuccinimide
0.197 g (0.0017 mol) was treated with 0.3 g (0.0017 mol) of Nt-butoxycarbonylglycine.
After reacting for 18 hours, the reaction mixture was diluted with 5 ml of tetrahydrofuran, and the filtrate was concentrated under reduced pressure to give a white solid, N-t-butoxycarbonylglycine-N-.
Hydroxysuccinimide ester was obtained. A solution of 1.137 g (0.00128 mol) of L-thyroxine sodium salt pentahydrate in 20 ml of methanol and 0.35 g (0.003 mol) of N-t-butoxycarbonylglycine N-hydroxysuccinimide ester.
was treated for 18 hours. Transfer the mixture to Bio-Rad AG
Pass through a 50W−X8 (H ⁺ type) ion exchange resin tube, treat with methanol, and concentrate under reduced pressure to obtain a white solid.
Gained 1.0g. After reacting 0.125 g (0.000134 mol) of a white solid with 3.0 ml of trifluoroacetic acid for 30 minutes, the acid was evaporated under reduced pressure, the residue was dissolved in 1.5 ml of N,N'-dimethylformamide, and the pH of the solution was made alkaline using triethylamine. And so. 0.0075 g (0.000159 mol) of 5-carboxyfluorescein N-hydroxysuccinimide ester was added to the resulting mixture.
A crude product was obtained after reacting for some time. This was purified by reversed phase thin layer chromatography using a methanol:water:acetic acid (75:25:0.5) mixture using the formula: An orange solid thyroxine-5-carboxyfluorescein conjugate was obtained. Example 6 1.03 g (0.010 mol) of gamma-aminobutyric acid and 2.49 g of N-benzyloxycarbonyloxysuccinimide in 10 ml of N,N'-dimethylformamide
(0.01 mol) was stirred at 22°C for 18 hours. The resulting clear solution was concentrated and water was added to the residue to form an oil that became white crystals. The dried residue was purified using a silica gel tube and dichloromethane: methanol (95:
5) Elution with the mixture yielded gamma(benzyloxycarbonylamino)butyric acid. Gamma (benzyloxycarbonylamino)
0.237g (0.001mol) of butyric acid in 2ml of dichloromethane
N,N'-dichlorohexylcarbodiimide in
0.206 g (0.001 mol) and 0.135 g (0.0012 mol) N-hydroxysuccinimide at 22° C. for 2 hours. Filter the reaction mixture and concentrate the liquid under reduced pressure to obtain gamma (benzyloxycarbonylamino).
The succinimide ester of butyric acid was obtained as an oily residue. This was reacted for 18 hours with 0.889 g (0.001 mol) of L-thyroxine sodium salt pentahydrate in 5 ml of methanol. The reaction mixture was transferred to Bio-Rad AG.
It was passed through a 50W-X8 (H ⁺ type) ion exchange resin tube and evaporated with methanol to give glassy white crystals. This was purified using silica gel tube chromatography and dichloromethane:methanol (9:
1) The mixture was eluted to give a white solid. To 0.25 g (0.000025 mol) of this white solid was added 0.4 ml of acetic acid containing 30% hydrobromic acid over 30 minutes. Addition of excess diethyl ether precipitated the hydrobromide salt. After washing and drying this, 5-carboxyfluorescein N- in 0.4 ml of N,N'-dimethylformamide was prepared in the presence of triethylamine.
Hydroxysuccinimide ester 0.25g
The product obtained by reacting with (0.00053 mol) for 16 hours was purified using reverse phase thin layer chromatography using a methanol:water:acetic acid (70:30:0.5) mixture using the formula: An orange solid thyroxine-5-carboxyfluorescein conjugate was obtained. As mentioned above, the tracer of the present invention is an effective reagent for use in fluorescence polarization immunoassays. The following example demonstrates the suitability of the tracer of the invention in immunoassays using fluorescence polarization. This test is carried out according to the following general method: (1) A known amount of test or standard serum is placed in a test tube and diluted with a buffer solution. (2) Add a known concentration of the tracer of the invention, optionally containing a surfactant, to each tube. (3) Add a known concentration of antiserum to the tube. (4) Incubate the reaction mixture at room temperature. (5) Measure the amount of tracer bound to the antibody by fluorescence polarization as a measure of the amount of ligand in the sample. Example 7 Thyroxine Test A Required materials: (1) PH7.5BGG buffer consisting of 0.1M sodium phosphate solution containing 0.01% bovine gamma globulin and 0.01% sodium azide. (2) A tracer consisting of the thyroxine carboxyfluorescein derivative prepared in Example 2 at a concentration of approximately 60 nM in BGG buffer. (3) Antiserum consisting of the sheep antiserum raised against thyroxine appropriately diluted in BBG buffer. (4) Human serum samples or other biological fluids containing thyroxine. (5) Serum denaturing agents - 8M urea in water, 3% sodium dodecyl sulfate, 1% dithioerythritol, 50mM ascorbic acid, 2mM sodium editate. (6) 10 x 75 mm glass culture tube used as a cube. (7) A fluorometer for measuring fluorescence polarization with an accuracy of ±0.001 units. B Test method 1 50μ of the standard or unknown sample was added to 50μ of serum denaturing agent in a test tube. The tube containing the sample was stoppered and the contents were mixed. 2 Pipette 29μ of the denatured sample into a dilution container containing 25ml of pretreatment reagent and 25ml of antiserum.
20μ of the sample was diluted in 500μ of BGG buffer and placed in each tube. Next, dilute sample 175μ
Pipette into the container and then
805μ of BGG buffer was taken. At this point, serum base fluorescence was called. 3. Pipette 75μ of diluted sample, 25μ of tracer and 780μ of BGG buffer into a test tube and add tracer. 4. Mix the contents of all the khvets well.35
The cells were incubated at ℃ for 4 minutes. 5. Fluorescence polarization was read using a fluorometer corrected against the serum fluorescence base and a standard curve prepared for determining unknown values. A series of results for serum standards containing thyroxine at concentrations from C 0 to 24 μg/dl are shown below. Each concentration is 2
This is the average value of multiple measurements. Thyroxine concentration (μg/dl) Polarization 0 0.223 3 0.209 6 0.197 12 0.173 18 0.155 24 0.143 The polarization of fluorescent light appears to decrease normally as the thyroxine concentration increases, forming a standard curve. Unknown samples processed in a similar manner can be quantified by comparing them to a standard curve. The above method was used to analyze the patient's serum, and the results were corrected by a radioimmunoassay (Abbott's T ₄ -PEG test). A correction coefficient of 0.0962 was obtained. As is clear from the above results, the tracer of the present invention is a useful reagent in fluorescence polarization immunoassays. In addition to the above properties, the tracers of the present invention have a high degree of thermal stability, a high degree of binding polarization, a high quantum yield, and are relatively easy to produce and purify. In addition to being useful as reagents in fluorescence polarization immunoassays, the thyroxine carboxyfluorescein derivatives of the present invention are also useful in fluorescence polarization assays for determining unsaturated thyroxine binding protein localization ("T uptake") by the method of the following example. Useful as a tracer. Example 8 A Reagent 1 Pretreatment Solution - 0.15% sodium dodecyl sulfate, 0.564M triethylenediamine (DABCO) and 0.1% sodium azide in 0.1M sodium phosphate buffer (PH 7.25). 2 T ₄ - Fluorescent Saint Tracer - 0.005
Compound 4 made in Example 5 is used at a concentration of 2.4 x 10 ^-7 M in 0.1 M sodium phosphate buffer containing % sodium dodecyl sulfate, 0.1% bovine gamma globulin and 0.1% sodium azide. 3 T-uptake caliber-0,
Sheep anti-T antiserum in a 4% human serum matrix with uptake values of 0.5, 1.0, 1.5, 2.0 and 2.5. An uptake value of 10 corresponds to the T uptake of normal serum. 4 Dilution buffer - 0.1% bovine gamma globulin
0.1M sodium phosphate solution containing 0.1% sodium azide. All polarized fluorescence measurements were performed using a polarization spectrofluorometer (Abbott TD _x ^TM Fluorescence Polarimeter). B Test method 1 A mixed solution in which 1μ of the unknown sample was added to 25μ of the pretreatment solution was adjusted to 1μ by adding dilution buffer.
The resulting test solution was mixed and the polarized fluorescent substrate was measured. 2 Unknown test 2nd 1μ to the test solution of step 1,
25μ of pre-treatment solution and 25μ of _T4 fluorescein tracer were added, and finally buffer was added to make the volume 2ml. The resulting solution was mixed and the polarized fluorescence was measured. 3. The polarized fluorescence intensity due to tracer binding was obtained by subtracting the polarized fluorescence intensity of the substrate from the final polarized fluorescence intensity of the mixed solution. 4 The obtained polarization value is proportional to the T uptake of each sample. 5 Compare the fluorescence polarization of the sample with a standard curve created using a calibrator with a known T uptake value to determine the T uptake value. Although specific modifications of the invention have been described, it will be obvious that various alternatives, changes and modifications may be made without departing from the spirit and scope of the invention, and similar embodiments are encompassed by the invention. As such, these details should not be construed as limiting the invention.

Claims (1)

[Claims] 1 Formula: or (In the above formula, T represents a group [formula], where n is an integer from 1 to 8, and R represents at least one epitope in common with the ligand so that it can be specifically recognized by a common antibody.) represents a ligand homologue with )
A fluorescent polarization immunoassay reagent comprising the compound represented by or a biologically acceptable salt thereof. 2. The reagent according to claim 1, wherein R is a thyroxine homolog. 3 R is the formula: The reagent according to claim 2. 4. The reagent according to claim 3, wherein n is 2 to 4. 5. The reagent according to claim 4, wherein n is 3. 6 Formula the sample: or (wherein T represents a group [formula], n is an integer from 1 to 8, and R represents at least one epitope common to the ligand so that it can be specifically recognized by a common antibody. After mixing with a tracer consisting of a compound represented by (representing a ligand homolog) or a biologically acceptable salt thereof and an antibody that can specifically recognize the above-mentioned ligand and the above-mentioned tracer, The above method for measuring ligands in a sample, which comprises measuring the amount of tracer bound to the antibody by fluorescence polarization as a measure of the amount of ligands. 7. The method according to claim 6, wherein R has a molecular weight of 50 to 4000. 8. The method of claim 7, wherein R is a thyroxine congener. 9 R is the formula: 9. The method according to claim 8. 10. The method according to claim 6 or 9, wherein n is an integer from 2 to 4. 11. The method of claim 10, wherein n is 3.